LOCUS       sequence-453625-        6832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TurboID-FRB-NLS fusion from HIS3 promoter; yeast BioID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6832)
  AUTHORS   Pfannenstein J, Tyryshkin M, Gulden ME, Doud EH, Mosley AL, Reese JC
  TITLE     Characterization of BioID tagging systems in budding yeast and 
            exploring the interactome of the Ccr4-Not complex.
  JOURNAL   G3 (Bethesda). 2024 Sep 13:jkae221. doi: 10.1093/g3journal/jkae221.
  PUBMED    39271111
REFERENCE   2  (bases 1 to 6832)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "G3 
            (Bethesda). 2024 Sep 13:jkae221. doi: 10.1093/g3journal/jkae221."
COMMENT     Sequence Label: pRS416-HIS3p-TID-FRB-NLS-HA3
FEATURES             Location/Qualifiers
     source          1..6832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     551..570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(789..893)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    930..1433
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     1474..1492
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1530..1552)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1652..1671
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1695..1910
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             1911..2714
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(2845..3300)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2982..3003)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3194..3213
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3426..3448
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3440..3457
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3441..3457
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3467..3486
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3467..3485
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3904..4091
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             5113..5391
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             5401..5421
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5422..5511
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(5560..5580)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5560..5578)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5599..5615)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5599..5615)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5612..5634)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5623..5639
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5647..5653,5654..5671,5672..5677))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5692..5713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5830..5847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6001..6589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6081..6100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6760..6832,1..719,720..788))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
       61 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
      121 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
      181 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
      241 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
      301 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
      361 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
      421 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
      481 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
      541 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
      601 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      661 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      721 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      781 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      841 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      901 ccccgaaaag tgccacctgg gtccttttca tcacgtgcta taaaaataat tataatttaa
      961 attttttaat ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa
     1021 atagtttttg ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct
     1081 tttatcttgc tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta
     1141 gaagaccaca cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata
     1201 tctatttaat ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa
     1261 attttttaaa cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat
     1321 ttactttcta aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa
     1381 attattccat cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atccgtctaa
     1441 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     1501 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     1561 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     1621 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     1681 caccatacca ccttttcaat tcatcatttt ttttttattc ttttttttga tttcggtttc
     1741 cttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca
     1801 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat
     1861 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag
     1921 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata
     1981 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat
     2041 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata
     2101 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt
     2161 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc
     2221 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg
     2281 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac
     2341 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat
     2401 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg
     2461 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg
     2521 tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg
     2581 tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg
     2641 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg
     2701 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta
     2761 gagcttcaat ttaattatat cagttattac cctatgcggt gtgaaatacc gcacagatgc
     2821 gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta aaattcgcgt
     2881 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     2941 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3001 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3061 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3121 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3181 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3241 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3301 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     3361 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     3421 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     3481 atagggcgaa ttgggtaccc attcataagt ttcagagcaa tgtccttatt ctggaacttg
     3541 gatttatggc tcttttggtt taatttcgcc tgattcttga tctcctttag cttctcgacg
     3601 tgggcctttt tcttgccata tggatccgct gcacggtcct gttccctagc atgtacgtga
     3661 gcgtatttcc ttttaaacca cgacgctttg tcttcattca acgtttccca ttgttttttt
     3721 ctactattgc tttgctgtgg gaaaaactta tcgaaagatg acgacttttt cttaattctc
     3781 gttttaagag cttggtgagc gctaggagtc actgccaggt atcgtttgaa cacggcatta
     3841 gtcagggaag tcataacaca gtcctttccc gcaattttct ttttctatta ctcttggcct
     3901 cctctagtac actctatatt tttttatgcc tcggtaatga ttttcatttt tttttttcca
     3961 cctagcggat gactcttttt ttttcttagc gattggcatt atcacataat gaattataca
     4021 ttatataaag taatgtgatt tcttcgaaga atatactaaa aaatgagcag gcaagataaa
     4081 cgaaggcaaa ggaagcttaa aatggcaaga gatccgcctg tggccacgat gaaagataac
     4141 acggttccct taaaattgat agcccttctt gccaatggtg agtttcattc cggggaacaa
     4201 cttggcgaaa ctctgggaat gagcagggca gcgattaaca agcatatcca aaccttgaga
     4261 gactggggag tagatgtgtt caccgtgcct ggtaaaggtt attctttgcc ggaacctatc
     4321 cctctattaa atgcgaaaca gatactagga caactggacg gtggtagtgt tgccgtattg
     4381 ccggttgttg actcaaccaa tcaatacttg ctggacagaa ttggcgaact taaaagcggt
     4441 gatgcgtgta ttgcagaata tcagcaagca gggagaggct caagaggtag gaaatggttt
     4501 tcaccttttg gcgccaatct ttacttaagt atgttctggc gtttgaagag agggcccgcg
     4561 gctattggtt taggtcccgt tatcgggata gttatggctg aagcgctacg taaacttggg
     4621 gccgacaagg tcagagtgaa gtggcctaat gatctatact tgcaagatag aaagctagcg
     4681 ggtatcctgg tggagctagc gggaatcaca ggggacgctg cccaaatagt aatcggtgct
     4741 ggtataaatg tcgcaatgcg tagagtcgag gaatcagtgg tgaaccaggg ctggatcacg
     4801 ttgcaagagg cgggcataaa cctggaccgt aacacccttg cagcgaccct aataagagaa
     4861 ctacgtgctg cattggagct ttttgagcag gagggcctgg ctccgtacct acccaggtgg
     4921 gagaaattag acaatttcat aaaccgtcca gtcaaactaa taattggaga taaggagatt
     4981 ttcggtattt ctcgtgggat agacaagcag ggagcgttgt tattagaaca ggatggtgtc
     5041 attaaacctt ggatgggcgg tgaaattagt cttcgtagtg ccgaaaaggc tagcggttct
     5101 ggtggtgcta gcatcctctg gcatgagatg tggcatgaag gcctggaaga ggcatctcgt
     5161 ttgtactttg gggaaaggaa cgtgaaaggc atgtttgagg tgctggagcc cttgcatgct
     5221 atgatggaac ggggccccca gactctgaag gaaacatcct ttaatcaggc ctatggtcga
     5281 gatttaatgg aggcccaaga gtggtgcagg aagtacatga aatcagggaa tgtcaaggac
     5341 ctcctccaag cctgggacct ctattatcat gtgttccgac gaatctcaaa gctcgagcca
     5401 ccaaaaaaga aaagaaaagt ttacccatac gatgttcctg actatgcggg ctatccgtat
     5461 gacgtcccgg actatgcagg atcttatcca tatgacgttc cagattacgc tgcttgatct
     5521 agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc
     5581 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5641 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5701 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5761 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5821 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5881 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5941 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6001 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6061 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tc
//
